Beta-Hydroxydecanoyl thioester dehydrase ("dehydrase") is the bacterial enzyme which functions at the branch point in the biosynthesis of saturated and unsaturated fatty acids under anaerobic conditions. Dehydrase catalyzes the following reversible reactions: dehydration of (3R)-3-hydroxydecanoyl thioester (in vivo, acyl carrier protein is the thiol) to E-2-decenoyl thioester; and isomerization of E-2-decnoyl thioester to Z-3-decenoate. "One base" and "two base" mechanisms have been proposed, and while sound evidence exists for an active site histidine, the identity of the "second "base" (if there is one) and the overall mechanism remain to be defined. Thioesters of 3-decynoic acid are "suicide substrate" inhibitors of dehydrase. The details of the inactivation process, which requires dehydrase-mediated isomerization to 2,3-decadienoyl thioester, and its relationship to dehydrase's normal mechanism of action are unclear. Previous research from this laboratory has been directed toward answering the one base-two base question by determining the normal stereochemical mode of action of dehydrase with particular emphasis on whether the reactions involve groups on the same or different faces of the substrate molecule. We now propose to investigate the regiochemistry and stereochemistry of dehydrase inactivation by 3-decynoic acid thioester. Specifically, we propose to a) determine unambiguously the position of the carbon-carbon double bond in the inactivator portion of the inhibited enzyme by synthesizing 13C -labeled inhibitor and analyzing the inactivated enzyme by 13C NMR; b) uncover the stereochemistry of the protonation in the alkylation of enzyme by 2,3-decadienoyl thioester (by synthesizing tritiated inhibitor followed by chemical degradation and enzymatic analysis of label position in the inactivated enzyme); and c) determine by a combination of chemical and physical methods the stereochemistry of the carbon-carbon double bond in the enzyme-alkylated inhibitor.